1. Field of the Invention
The invention relates to a device for carrying out, in particular, quantitative fluorescence-marked affinity tests by means of evanescent field excitation. This may involve a wide variety of known biochemical assays of general receptor/ligand systems, such as antibody/antigen, lectin/carbohydrate, DNA or RNA/complementary nucleic acid, DNA or RNA/protein, hormone receptor, enzyme/enzyme cofactors, [sic] protein or protein A/immunoglobin [sic] or avidin/biotin. Preferably however, antibody/antigen systems are evaluated.
2. Description of the Prior Art
Fluorescence immunotests, or fluorescence immunosensors, use an antibody/antigen system and have long been used widely. They are used primarily to quantify an unknown amount of a particular chemical or biochemical substance in a liquid sample matrix. In this context, antibodies are bound selectively to the substance to be determined. The substance to be determined is referred to by the person skilled in the art as an antigen. In fluorescence immunotests, the analyte-specific antibodies are marked with a marking substance which is optically excited at a particular substance-specific wavelength .lambda..sub.ex and the fluorescent light with a different wavelength, which is generally longer, is used with a suitable detector with evaluation of the fluorescent light intensity. The use of evanescent field excitation when implementing such fluorescence immunotests, or respectively the fluorescence immunosensors, already belongs to the prior art. For example, a variety of solutions have already been described in WO 94/27137, by R. A. Badlay, R. A. L. Drake, I. A. Shanks, F. R. S., A. M. Smith and P. R. Stephenson in "Optical biosensors for immunoassays: fluorescence capillary-fill device", Phil. Trans. R. soc. Lund. B 316, 143 to 160 (1987) and D. Christensen, S. Dyer, D. Flowers and J. Herron, "Analysis of Exitation [sic] and Collection Geometries for Planar Waveguide Immunosensors", Proc. SPIE-Int. Soc. Opt. Eng. Vol. 1986, Fiber Optic Sensors in Medical Diagnostics, 2 to 8 (1993). However, the known solutions generally have the disadvantage that they require relatively great outlay in order for the light needed to generate the fluorescence to be coupled into an optical fiber or for the fluorescent light to be extracted, which form an essential part of hitherto customarily used devices.
Further, U.S. Pat. No. 3,939,350 describes a solution in which fluorescence immunoassays are carried out by means of evanescent field excitation.
In this case, light from a light source is directed at an angle through a prism onto an interface, so that total reflection takes place and the fluorescence caused in a sample can be measured with a detector. The entire sample volume is in this case accommodated in a sealed closed space, so that on account of the relatively large sample volume only diffusion-controlled end-point detection can be carried out and this is susceptible to error.
WO 90/05295 describes an optical biosensor system in which [lacuna] an elaborate optical system excitation light can be directed onto sensitive regions of a likewise elaborate channel system, through which the sample volume is fed by means of controlled valves and pumps, and the fluorescent light emerging through windows from the sensitive regions can be re-directed onto a detector with a view to intensity measurement. Besides the aforementioned disadvantageous complex and elaborate structure, this system requires, before and after a test is carried out, cleaning both of the pumps and of the entire channel system in order to preclude the possibility of subsequent measurement errors.
WO 90/06503 describes a sensor in which excitation light is directed at a suitable angle through an optically transparent substrate onto an interface to form an optically transparent buffer layer, over which an extra waveguide layer, to which the analytes to be determined are in turn bound, is applied.
The refractive index of the buffer layer is in this case lower than that of the substrate and of the waveguide. If a suitable choice is made for the angle of the excitation light, total reflection takes place at the substrate/buffer boundary layer [sic] and, by means of the resulting evanescent field, the excitation light is coupled into the waveguide lying over the buffer layer. The light coupled into the waveguide is guided by means of total reflection in the waveguide, and the resulting evanescent field is correspondingly employed for fluorescence excitation.
The sample may be accommodated in one or more cavities, the only restriction on the corresponding dimensioning of such a cavity being that its size permits the sample to be transported into the cavities by means of capillary force. After the sample has been taken in by the cavities, no further flow or movement of the sample takes place.
WO 89/09408 A1 discloses a similar solution, which once more uses the light source for the excitation light and the detector for the fluorescent light on the same side. The sample to be detected is accommodated in a cavity between a waveguide and a cover plate. Here [sic] again no further flow or movement of the sample takes place after the sample has been taken up.